BIODEGRADATION OF TRICLOSAN BY A TRICLOSAN-DEGRADING ISOLATE AND AN AMMONIA-OXIDIZING BACTERIUM A Thesis by FUMAN ZHAO Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE May 2006 Major Subject: Civil Engineering
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BIODEGRADATION OF TRICLOSAN BY A TRICLOSAN … fileiii ABSTRACT Biodegradation of Triclosan by a Triclosan-degrading Isolate and an Ammonia-oxidizing Bacterium. (May 2006) Fuman Zhao,
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BIODEGRADATION OF TRICLOSAN BY A TRICLOSAN-DEGRADING
ISOLATE AND AN AMMONIA-OXIDIZING BACTERIUM
A Thesis
by
FUMAN ZHAO
Submitted to the Office of Graduate Studies of Texas A&M University
in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
May 2006
Major Subject: Civil Engineering
BIODEGRADATION OF TRICLOSAN BY A TRICLOSAN-DEGRADING
ISOLATE AND AN AMMONIA-OXIDIZING BACTERIUM
A Thesis
by
FUMAN ZHAO
Submitted to the Office of Graduate Studies of Texas A&M University
in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
Approved by:
Chair of Committee, Kung-Hui Chu Committee Members, Timothy Kramer Hongbin Zhan Head of Department, David V. Rosowsky
May 2006
Major Subject: Civil Engineering
iii
ABSTRACT
Biodegradation of Triclosan by a Triclosan-degrading
Isolate and an Ammonia-oxidizing Bacterium. (May 2006)
Fuman Zhao, B.S., University of Tianjin, China
Chair of Advisory Committee: Dr. Kung-Hui Chu
Triclosan is incorporated in a wide array of medical and consumer products as
an antimicrobial agent or preservative. Disposal of these products transport triclosan into
wastewater and later into soils and surface waters. Due to incomplete removal of
triclosan in wastewater treatment plants, contamination of triclosan in the environment
has raised several concerns, including: (i) an aid to the development of cross-resistance
to antibiotics, (ii) the toxicity to ecological health, (iii) the formation of chlorodioxins
from triclosan and its metabolites. By using 14C-labeled triclosan, 14CO2 was observed in
activated sludge samples, suggesting that triclosan was biodegraded. However, little is
known about the microorganisms responsible for triclosan biodegradation in activated
sludge.
The goal of this study is to better understand biodegradation of triclosan in
activated sludge. Two specific objectives are: (i) isolating and characterizing triclosan-
degrading bacteria from activated sludge, (ii) characterizing the cometabolic degradation
of triclosan through an ammonia-oxidizing bacterium Nitrosomonas europaea.
iv
A triclosan-degrading strain, KCY1, was successfully isolated from the
activated sludge. The strain KCY1 completely degraded triclosan in three days when
OD600 was 0.4. Based on 16S rRNA analysis, the strain KCY1 has 97% similarity with
Phingomonas or Phingopyxis. Negative results of oxygenase activity assays suggested
that other enzymes rather than oxygenases might be responsible for the triclosan
biodegradation. Experiments using N. europaea showed that triclosan could be
cometabolized. In the presence of inhibitor for ammonia monooxygenase (AMO), N.
europaea was unable to degrade triclosan, suggesting that AMO might be responsible
for triclosan degradation. Triclosan appeared to competitively inhibit ammonia oxidation
by N. europaea. Results of this study showed that triclosan might be effectively
biodegraded by triclosan-degrading cultures, strain KCY1 and N. europaea.
v
ACKNOWLEDGEMENTS
I thank my committee members: Dr. Kung-Hui Chu, my committee chair, for
her guidance, advice, criticism and encouragement; Dr. Timothy Kramer for his support
and counsel; and Dr. Hongbin Zhan for his suggestions, availability and commitment.
I thank Dr. Chang-ping Yu for his friendly, insightful, detailed directions and
comments of my experiment conductions.
A special thank you goes to my family for their love, support, encouragement,
and patience, to my lab co-workers for their help, support, and friendship.
LIST OF TABLES………....………………………….……….…………………..….…ix
CHAPTER…..……………………………………………………………………………1
I INTRODUCTION……………………………………………………………1
II BACKGROUND.……………….…………………………………………4
Physical and Chemical Properties…..……………………………………4 Activity Mechanism and Effectiveness……..……………………………5 Toxicity……...……………………………………………………………7 Environmental Effects…...………………………………………………9 Regulation and History……...……………………………………..10 Degradation….…………………………………………………………..11 Ammonia-oxidizing Bacteria and Ammonia Monooxygenase……..…..14
Growth of Nitrosomonas europaea………...………………..………….18 Triclosan-degrading Consortium……..…….………………………….18 Isolation……...………………………………………………………….19 DNA Extraction and PCR Amplification……………………………….21 16S rRNA Sequence………….….………………………………….22 Tests of Triclosan Degradation by an Isolate……….…………………..23 Tests of Triclosan Degradation by N. europaea…………….………….24 Analytical Methods…..…………..………..……………………….25
vii
CHAPTER Page
IV TRICLOSAN-DEGRADING CONSORTIUM AND AN ISOLATE KCY1…..…………………………………………….26
Triclosan-degrading Consortium…...……………………....………26 Isolation of Triclosan-degrading Strain KCY1……………..……….28 Phylogenetic Analysis of KCY1..…………………………………….31 KCY1’s Ability to Use Triclosan as a Sole Carbon Source…..…….34
Oxygenase Activity Assays……………………………………….35
V BIODEGRADATION OF TRICLOSAN BY NITROSOMONAS EUROPAEA……….………………………………….37
Introduction…..………………………………………………………….37 Results and Discussion………………………………………………….38 Triclosan degradation by N. europaea........…………………..38 Effects of NADH on triclosan degradation……………….…….42
Competitive inhibition between ammonia and triclosan….….43 Discussion……………………………………………………….44
VI CONCLUSION AND FUTURE STUDIES……………………………….46
REFERENCES………………………………………………………………………….49
APPENDIX..………………………………………………………………………….…59
VITA ...………………………………………………………………………………….69
viii
LIST OF FIGURES
FIGURE Page
2.1 Triclosan chemical structure and molecular model...……………………………4
2.2 Proposed triclosan degradation pathways in presence of free chlorine…………12
2.3 Ammonia oxidation by N. europaea by enzymes AMO and HAO……………..16
3.1 Schematic procedures for isolating triclosan-degrading cultures………...…..20
4.1 Degradation of triclosan in four successive culture transfers.………..…..26
4.2 Biodegradation of triclosan by the fourth enrichment culture….………….……27
4.3 Isolated yellow and mucoid colony on R2A-triclosan agar plates.……………..28
4.4 Biodegradation of triclosan by using resting cells of strain KCY1…….………30
4.5 Agarose gel electrophoresis of PCR-amplified product DNA ……..…………..32
4.6 Phylogenetic relationships between strain KCY1 and other known bacteria….33
4.7 Degradation of triclosan by strain KCY1 growing in NMS-triclosan medium...34
5.1 Triclosan degradation by N. europaea with 2 mg/L triclosan.....………….….39
5.2 Triclosan degradation by N. europaea with 0.5 mg/L triclosan…...……….….39
5.3 Triclosan degradation by N. europaea in the presence of AMO inhibitor….…40
5.4 Nitrite production by N. europaea in the presence of AMO inhibito………...41
5.5 Ammonia oxidation by N. europaea in the presence of AMO inhibitor……...41
5.6 Triclosan degradation by N. europaea in the presence of formate..…………….42
5.7 Nitrite production over time by N. europaea in the presence of formate………43
5.8 Nitrite production by N. europaea in the presence or absence of triclosan.…..44
ix
LIST OF TABLES
TABLE Page
2.1 EC50 values for triclosan, phenol, and copper……………………...……………8
4.1 Recovery of chloride following triclosan degradation by strain KCY1..……….30
4.2 Bacteria with a high homology in the 16S rRNA sequence of strain KCY1…..32
4.3 Indole oxidation assay: Absorbance (A400) change in 90 min…………………..35
4.4 Naphthalene oxidation assay: Absorbance (A530) changes in 24 hours……..36
1
CHAPTER I
INTRODUCTION
Triclosan, an antimicrobial agent and preservative, has been widely used for
over 30 years since its first introduction into the health care industry in a surgical scrub
in 1972 (Glaser, 2004). Triclosan can block lipid biosynthesis by inhibiting the enzyme
enoyl acyl carrier protein reductase and may induce bacterial resistance development
(McMurry et al., 1998; Levy et al., 1999). Currently, Triclosan has been incorporated in
a broad array of personal care products (e.g. hand disinfecting soaps, medical skin
completed with CLUSTAL X software, and the bootstrap support values from 1000
replication are indicated at branch nodes. A phylogenetic tree was plotted by using a
software Tree View.
Tests of Triclosan Degradation by an Isolate
Indole oxidation analysis: Dioxygenase activity in cells was measured in term
of the oxidation of indole into cis-indole-2,3- dihydrodiol, indoxyl, and finally indigo.
The assays were performed as described by Jenkins and Dalton (Jenkins and Dalton,
1985). The isolate was pregrown in 10% TSB medium for two days before harvested
through centrifuge at 10000xg rpm and 4 °C for 30 min. After washing with 0.05 M
KH2PO4 buffer solution, the pellets were resuspended with cell solution (containing 50
ml DI water, 10 ml pure glycerol, 10 ml filtered alcohol, 10 ml of 0.05 M KH2PO4, 10 ml
of 100 mM DTT and 10 ml of 0.05 M NADH). French pressure was used to break the
cells. The experimental samples were prepared with a volume of 3 ml including 0.15 ml
of 0.5 KH2PO4 buffer solution, 3 μl of 0.1 M FeSO4•7H20, 0.02 ml of 0.05 M NADH,
0.3 ml of 2 mM indole and 2.53 ml cell suspension. Dioxygenase activity was
determined by measure the absorbance at A400 after 90 minutes of incubation. The
absorbance was detected by a Pierce spectrophotometer. A blank containing all
ingredients except indole was used as background absorbance.
Naphthalene oxidation assays: The activity of monooxygenase was measured by
naphthalene oxidation assay as described by Chu and Alvarez-Cohen (Chu and Alvarez-
Cohen, 1998). The assay is based on that monooxygenase can oxidize naphthalene to 1-
or 2-naphthol which reacts with tetrazotized o-dianisidine to form a purple naphthol-
24
diazo complex. The quality of naphthol-diazo complex is measured through absorbance
at 530nm. Isolate was pregrown with 10% TSB medium to optical density (600 nm) 0.2.
Glass vials were amended with 1 ml cell suspension, 1 ml of saturated naphthalene stock
solution and 1 ml of 20 mM sodium formate. The amended vials were incubated at 30
°C, at 160 rpm for 1 hour. After adding 100 μl of the freshly made 0.2% (w/v)
tetrazotized o-dianisidine, the absorbance (530 nm) was measured promptly in two
minutes by using a Pierce spectrophotometer. Blank controls containing only cells and
formate (no naphthalene) were employed. Differences in absorbance between blanks
and samples were determined after incubation for 24 hours.
Tests of Triclosan Degradation by N. europaea
Experiments were conducted to examine triclosan degradation by N. europaea
at two different triclosan concentrations (0.5 mg/L and 2.0 mg/L). The flasks contained
300 ml of acetone-free growth medium that was prepared as follows: First, adding a
known amount of triclosan-acetone stock solution into empty flasks; After acetone
completely evaporated, adding the N. europaea growth medium and waiting for at least
2 days to allow triclosan to completely dissolve in the growth medium; and finally
adding cell suspension prepared previously. Additional sets of flasks were amended with
10 mg/L allylthiourea as an AMO inhibitor (Rasche et al, 1991), and 20 mM sodium
formate as an oxygenase reducing power. Positive controls (without triclosan) were used
to assess ammonia oxidation activities of N. europaea. Initial protein concentrations
were measured by using a BCA Protein Assay Reagent Kit (Pierce, Rockford, IL).
Concentrations of triclosan, ammonia, and nitrite were monitored over time and
25
measured by GC-MC, IC, and ammonia-selective electrode probe, respectively. Samples
were filtered with Millex-GP filter unit (0.22 μm pore size) before ammonia and nitrite
analyses.
Analytical Methods
Concentrations of triclosan were determined by GC-MS analysis. Triclosan in
liquid samples was extracted with ethyl ether overnight, and the extracts were derivitized
with 50 ml DMF and 450 ml BSTFA. The derivitized samples were injected into a
Hewlet Packard 5890 Series II Gas Chromatograph and detected by a Hewlett Packard
5972 Mass Spectrometer (Lab Extreme Inc., Kent city, MI). The GC was equipped with
a HP-5MS capillary column (30 m x 0.25 mm i.d., df: 0.25 um) purchased from Agilent
(Wilmington, DE, USA). Helium (purity 99.999%) was used as carrier gas at a constant
flow of 1.2 ml/min. Automatic injections were performed in splitless flow (split ratio
equals 1.00:1) 1.2 ml/min. The each injected volume was 1 μl. The GC oven temperature
was operated as follows: starting at 80 °C, increased with a rate of 30 °C/min to 280 °C
and held for 3 minutes, finally to 300 °C and held for 3 minutes. The GC-MS interface
temperature was 290 °C. Mass spectra were obtained in selected ion monitoring (SIM)
mode (600 eV) at the two mass to charge values 200 and 360 for triclosan. The
monitoring peak areas were converted to triclosan concentrations by comparing them
with the standard curves constructed from standard solutions.
26
CHAPTER IV
TRICLOSAN-DEGRADING CONSORTIUM AND AN ISOLATE KCY1
Triclosan-degrading Consortium
After depletion of added triclosan, a known amount of cell suspension was
transferred to a new glass bottle containing NMS medium and 5 mg/L of triclosan. Four
successive transfers were conducted (Fig. 4.1). The ratio of cell suspension to NMS
medium for each transfer was 40 mL/80 mL (v/v). Despite triclosan was degraded in
transfer bottles, no significant changes in turbidity were observed.
Fig. 4.1. Degradation of triclosan in four successive culture transfers.
27
An additional set of the fourth enrichment culture was used to estimate initial
degradation rate of triclosan (Fig. 4.2).
0
1
2
3
4
5
6
0 2 4 6 8
Time (days)
Tric
losa
n C
onc.
(mg/
L)
10
sampleskilled controls
Fig. 4.2. Biodegradation of triclosan by the fourth enrichment culture. The volume ratio of cell suspension to NMS medium is 10 ml to 90 ml. Spiked triclosan concentration was 5 mg/L, and in aerobic condition. The bars indicate ranges of duplicate samples.
The initial specific degradation rate of triclosan was 0.17 nmol/min/mg of
protein, based on 11.2 mg protein/L measured in the fourth enrichment culture. Since
protein contents were not measured in the first three enrichment cultures, theoretical
calculations were conducted. The calculations were made by using 0.2 mg protein/L and
and known dilution factors employed during culture transfers. The protein content was
estimated by assuming 5 mg/L triclosan was used as a sole carbon source (70% of
substrate metabolized for biomass growth; 13% of protein content in biomass). The
28
calculated degradation rates for the first, second, and third enrichment cultures were
0.029 nmol/min/mg of protein, 0.047 nmol/min/mg of protein, and 0.076 nmol/min/mg
of protein, respectively. Therefore, triclosan-degrading consortium degrading capacity
increased through the enrichment processes.
Isolation of Triclosan-degrading Strain KCY1
After the fourth enrichment, cell suspension was used for isolation. Isolation of
triclosan-degrading cultures was conducted by streaking on NMS-triclosan agar plates,
after one time streaking, morphologically distinct colonies were selected and substreaked
on R2A-triclosan agar plates four times to check for the purity of strains. Three
presumptive triclosan-degrading colonies grew much faster on R2A-triclosan agar plates
than on NMS-triclosan agar plates. There three presumptive cultures showed
distinguishable colonies on R2A-triclosan agar plates: one is white and shiny, one is
opaque and whitish, and the third is yellow and mucoid (Fig. 4.3).
Fig. 4.3. Isolated yellow and mucoid colony on R2A-triclosan agar plates. The plates were incubated under aerobic conditions at 30 °C.
29
When streaking these three single colonies back onto NMS plates (no triclosan),
the white-and-shiny colony and the opaque-and-whitish colony were able to grow on the
plates. No growth of the yellow-and-mucoid colony was observed. This result
demonstrated that the white-and-shiny colony, the opaque-and-whitish colony could use
other carbon sources except triclosan or other than triclosan to satisfy their carbon and
energy requirements for their growths.
Experiments using resting cells were conducted to examine colonies’ ability to
degrade triclosan. Each isolate was pregrown with 10% TSB medium for 2 days and
harvested for experimental use. The cells were spun down and resuspended with NMS
medium to 0.4 of OD600. Experiments were conducted in 250 ml glass vials containing
100 ml cell suspensions and 5 mg/L of triclosan. The vials were incubated at 30 °C and
150 rpm. Concentrations of triclosan were measured over time. After 7 days of
incubation, no degradation of triclosan was observed in vials containing the white-and-
shiny colony and the opaque-and-whitish colony. Triclosan degradation was only
observed in vials containing the yellow-and-mucoid colony. About 80% of triclosan
added was degraded within 1 day. After 3 days, all the spiked triclosan concentration
almost approached zero (Fig. 4.4). Thus, only one strain of triclosan-degrading bacteria
was successfully isolated, and named as strain KCY1.
Because triclosan is a chlorinated organic compound, the chloride ion loss from
the parent compound can be used to evaluate if triclosan was fully mineralized by the
strain KCY1. If 5 mg/L triclosan is completely mineralized, it will release 1.84 mg/L of
30
chloride ion. The measured chloride ion concentration was 1.88 mg/L, suggesting that
triclosan was completely mineralized by the strain KCY1 (Table 4.1).
0
1
2
3
4
5
6
0 1 2 3 4 5 6 7Time (days)
Tric
losa
n C
onc.
(mg/
L)
sampleskilled controls
Fig. 4.4. Biodegradation of triclosan by using resting cells of strain KCY1. The bars indicate ranges of duplicate samples
Table 4.1 Recovery of chloride following triclosan degradation by strain KCY1. The initial triclosan concentration was 5 mg/L.
Initial Cl¯ concentration (mg/L) 18.87
Final Cl¯ concentration (mg/L) 20.75 Measurements
Produced Cl¯ concentration (mg/L) 1.88
% of Cl¯ in triclosan 36.8% Theoretical
prediction* Cl¯ production (mg/L) 1.84
* 3 moles of chloride ion released per 1 moles of triclosan mineralized.
31
Phylogenetic Analysis of Strain KCY1
After PCR amplification of 16S rRNA of strain KCY1, the PCR products were
separated from genomic DNA in agarose gel electrophoresis. As shown in Fig. 4.5, the
16S rRNA fragment size was consistent with the expected size (~1400 bp) determined
by FluorChem 5500 (Alpha Innotech, San Leandro, CA), suggesting that 16S rRNA was
successfully amplified and separated.
According to 16S rRNA sequence analysis, isolate KCY1 belongs to a member
of the genus Sphigomonas within α-proteobacteria or the genus of Sphigopyxis within α-
proteobacteria (Table 4.2). The phylogenetic tree (Fig. 4.6) shows the relationship
between strain KCY1 and the most closely related bacteria that was deposited in
GeneBank database. Strain KCY1 shows 97% similarity to two well-studied organic
contaminant degraders, phenanthrene-degrading bacterium and Sphingomonas sp. DB-1.
The known triclosan-degrading isolate Sphingomonas sp. Rd1 (Hay et al., 2001) has
91% similarity to strain KCY1. Interestingly, five estrogen-degrading bacteria KC11,
KC9, KC10, KC14 and KC8 (isolated in our lab) were found to have high similarities
(96%, 95%, 95%, 92%, and 92% respectively) to strain KCY1.
32
Fig. 4.5, Agarose gel electrophoresis of PCR-amplified product DNA. Lane A shows the negative control without PCR product template; Lane B shows the sample with PCR product template; Lane C shows 1 Kb plus DNA ladder.
Table 4.2 Bacteria with a high homology in the 16S rRNA sequence of strain KCY1.
Fig. 4.6. Phylogenetic relationships between strain KCY1 and other known bacteria. ¹previously isolated triclosan-degrading strain; ² DDT-degrading bacteria; ³ alkaliphilic microcystin-degrading bacteria. Bootstrap support values from 1000 replicates are indicated at branch nodes.
34
KCY1’s Ability to Use Triclosan as a Sole Carbon Source
Additional experiment was conducted to determine whether strain KCY1 can
grow on triclosan. When strain KCY1 was grown in NMS medium with 5 mg/L
triclosan (not experiencing enrichment through 10% TSB medium), a very long lag
phase appeared before triclosan degradation occurred (Fig. 4.7). After 33 days of
incubation, about 80% of spiked triclosan was degraded. However, no significant
increase in optical density or measurable protein content was observed. Despite that
triclosan was the only carbon source available for the pure culture KCY1, this result was
insufficient to determine whether KCY1 can use triclosan as a sole carbon and energy
source.
0
1
2
3
4
5
6
0 5 10 15 20 25 30 35
Time (days)
Tric
losa
n C
onc.
(mg/
L)
samples
killed controls
Fig. 4.7. Degradation of triclosan by strain KCY1 growing in NMS-triclosan medium. Triclosan with concentration 5 mg/L was contained as a sole carbon source.The bars indicate ranges of duplicate samples
35
Oxygenase Activity Assays
Indole oxidation assay and naphthalene oxidation assay were employed to
measure non-specific di- and mono- oxygenases activities in strain KCY1. Positive
color change indicates the presence of enzyme activity. No color and absorbance
changes were observed in indole oxidation assay (Table. 4.3) and in naphthalene
oxidation assay (Table. 4.4).
Table 4.3 Indole oxidation assay: Absorbance (A400) changes in 90 min
The negative results of di- and mono-oxygenase activity assays suggested that
non-specific oxygenases were not present in strain KCY1.
37
CHAPTER V
BIODEGRADATION OF TRICLOSAN BY NITROSOMONAS EUROPAEA
Introduction
Organic compounds can be biotransformed by microorganisms through two
different mechanisms: growth-linked (using these compounds as growth substrates) and
non-growth-linked (using these compounds as cometabolic substrates). In the growth-
linked processes, biodegradation occurs when the organism consumes the organic
compound as a primary substrate to satisfy its energy and organic carbon needs. On the
other hand, non-growth-linked processes are a fortuitous transformation of an organic
compound by enzymes or other biomolecules that are produced by microorganisms for
other purposes. The microorganisms obtain no obvious or direct benefit through
cometabolic degradation. In many case, cometabolic metabolites are harmful to
microorganisms (Ward et al., 1997).
Extended studies show that microorganisms with non-specific monooxygenase
or dioxygenase have the potential to initiate the cometabolic transformation (Ward et al.,
1997). Ammonia monooxygenase (AMO) is a type of non-specific monooxygenase
enzyme. Previous researches revealed that the physiological role of AMO is to
metabolize growth-supporting substrate ammonia and simultaneously cometabolize a
large range of non-growth-supporting substrates including hydrocarbon organic
compounds, halogenated aliphatic organic compounds, and halogenated aromatic
organic compounds (Vannelli et al, 1990; Keener and Arp, 1994). Triclosan belongs to a
38
chlorinated aromatic organic compound, and it is possible to detect its cometabolic
transformation by Nitrosomonas europaea under proper conditions.
This study will address several questions concerning with the activity of AMO.
The first question is to determine whether triclosan can be cometabolically transformed
by an ammonia-oxidizing bacterium, N. europaea. The second question is to determine
whether AMO plays an important role for the transformation, if degradation of triclosan
by N. europaea observed. If AMO is found to involve in triclosan degradation, questions
such as competitive inhibition and limitation of reducing will be addressed.
Results and Discussion
Triclosan degradation by N. europaea
Ammonia-oxidizing bacterium N. europaea was capable of degrading triclosan at
concentrations of 2 mg/L and 0.5 mg/L ( Fig. 5.1 and Fig. 5.2). These experiments were
conducted with the same initial cell concentration (13 mg of protein /L) and ammonia
concentration (700 mg-N/L). No triclosan degradation was observed in killed controls
or in the presence of allylthiourea, an AMO inhibitor (Fig. 5.3).
39
0
0.5
1
1.5
2
2.5
0 1 2 3 4 5Time (days)
Tric
losa
n C
onc.
(mg/
L)
samplesinhibitionskilled controls
Fig. 5.1. Triclosan degradation by N. europaea with 2 mg/L triclosan. Initial cell protein concentration was 13 mg/L, and initial ammonia concentration was 700 mg-N/L. Experiment was conducted under aerobic conditions at 30 °C. The bars indicate ranges of duplicate samples .
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3 4 5
Time (days)
Tric
losa
n C
onc.
(mg/
L)
Sampleskilled controlsinhibitions
Fig. 5.2. Triclosan degradation by N. europaea with 0.5 mg/L triclosan. Initial cell protein concentration was 13 mg/L, and initial ammonia concentration was 700 mg-N/L. Experiment was conducted under aerobic condition at 30 °C. The bars indicate ranges of duplicate samples
40
Similarly, neither production of nitrite (Fig. 5.4), nor depletion of ammonia
(Fig. 5.5) were observed, indicating that allylthiourea completely inhibited the activity
of AMO enzyme.
0
0.5
1
1.5
2
2.5
0 1 2 3 4
Time (days)
Tric
losa
n C
onc.
(mg/
L)
5
Triclosan-2mg/LTriclosan-0.5mg/L
Fig. 5.3. Triclosan degradation by N. europaea in the presence of AMO inhibitor. Initial triclosan concentration 2.0 mg/L and 0.5 mg/L, initial cell protein concentration was 13 mg/L, and initial ammonia concentration was 700 mg-N/L. Experiment was conducted under aerobic condition at 30 °C.
41
0
10
20
30
40
50
0 1 2 3 4Time (days)
Nitr
ite C
onc.
(mg/
L)
5
Triclosan-2mg/LTriclosan-0.5mg/L
Fig. 5.4. Nitrite production by N. europaea in the presence of AMO inhibitor. Initial triclosan concentration 2.0 mg/L and 0.5 mg/L, initial cell protein concentration was 13 mg/L, and initial ammonia concentration was 700 mg-N/L. Experiment was conducted under aerobic condition at 30 °C.
0
100
200
300
400
500
600
700
800
0 1 2 3 4 5Time (days)
Am
mon
ia C
onc.
(mg/
L)
Triclosan-0.5mg/LTriclosan-2mg/L
Fig. 5.5. Ammonia oxidation by N. europaea in the presence of AMO inhibitor. Initial triclosan concentration 2.0 mg/L and 0.5 mg/L, initial cell protein concentration was 13 mg/L, and initial ammonia concentration was 700 mg-N/L. Experiment was conducted under aerobic condition at 30 °C.
42
Effects of NADH on triclosan degradation
Experiments were conducted to examine the effects of NADH on triclosan
degradation by N. europaea. Sodium formate was provided as an external NADH source.
Interestingly, no effects of NADH on triclosan degradation were observed (Fig. 5.6).
Furthermore, the production of nitrite was not affected by the addition of formate (Fig.
5.7).
0
0.5
1
1.5
2
2.5
0 1 2 3 4 5Time (days)
Tric
losa
n C
onc.
(mg/
L)
w/ formatew/o formate
Fig. 5.6. Triclosan degradation by N. europaea in the presence of formate. Initial triclosan concentration 2.0 mg/L, initial cell protein concentration was 13 mg/L, and initial ammonia concentration was 700 mg-N/L. Experiment was conducted under aerobic conditions at 30 °C.
43
0
20
40
60
80
100
0 10 20 30 40 50 60 70 80Time (hours)
Nitr
ite C
onc.
(mg/
L)
w/ formate
w/o formate
Fig. 5.7. Nitrite production over time by N. europaea in the presence of formate. Initial triclosan concentration 2.0 mg/L, initial cell protein concentration was 13 mg/L, and initial ammonia concentration was 700 mg-N/L. Experiment was conducted under aerobic conditions at 30 °C.
Competitive inhibition between ammonia and triclosan
Previous studies with N. europaea have demonstrated that alternative substrates
for AMO could often exert an inhibitory effect on ammonia oxidation rates (Juliette et
al., 1993; Keener and Arp, 1993). This inhibitory effect occurred because of competitive
interactions between ammonia and the alternative substrate for AMO. The effects of this
competition could be most easily detected by examining accumulation rates of nitrite. In
this experiment, ammonia oxidation and triclosan degradation revealed an inhibitory
competition between each other (Fig. 5.8).
44
0
50
100
150
200
250
300
350
0 20 40 60
Time (hours)
Nitr
ite C
onc.
(mg/
L)
80
w/ triclosan
w/o triclosan
Fig. 5.8. Nitrite production by N. europaea in the presence or absence of triclosan. Initial triclosan concentration was 2.0 mg/L. Initial cell concentration was 13 mg protein /L. Initial ammonia concentration was 700 mg-N/L. Experiment was conducted under aerobic conditions at 30 °C.
Discussion
Previous studies have reported wide substrate ranges of AMO enzyme produced
by N. europaea (Hyman et al. 1988; Rasche et al., 1991). However, rarely the
chlorinated aromatic compounds have been shown to be degraded by AMO (Keener and
Arp, 1994). This experiment showed that triclosan could be degraded completely
through AMO oxidizing-activity, suggesting that AMO might be capable of